The overall goal of this proposal is to rationally design "loss of function" phospholamban (PLN) mutant candidates for the purpose of ameliorating the onset of cardiomyopathy by recombinant adeno-associated virus (rAAV) gene therapy. PLN is the endogenous inhibitor of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), the integral membrane enzyme responsible for 70% of the Ca2+ shuttling into the SR, inducing cardiac muscle relaxation in humans. Dysfunctions in SERCA:PLN interactions have been implicated as having a role in cardiac disease. The pathway to innovating genetic treatments for dysfunctions of SERCA and PLN interactions has recently become a new interest in the field due to the success of S16E, a pseudo-phosphorylated form of PLN which successfully reduced the progression of the cardiac failure in murine models and large animals. It has been found that the structural dynamics of PLN can be tuned to result in a directed functional effect on SERCA , and this study aims to innovate other candidates like S16E for use in gene therapy in treatment of heart failure. This proposal will concentrate on several key components of the domain-oriented rational design of PLN mutant species, and focus on these specific aims: AIM 1: Define the role of domain communication in PLN's ability to inhibit SERCA. AIM 2: Structural-dynamic-functional characterization of dual pseudo-phosphorylated PLN (S16E/T17E). AIM 3: Elucidate the significance of "superinhibitors" of PLN. New PLN mutant species will be developed using site-directed mutagenesis and expressed in Escherichia coli and purified using established methods. To characterize these new PLN mutants in their unphosphorylated and phosphorylated states, the structural-dynamics will be studied using solution-state nuclear magnetic resonance (NMR) spectroscopy in dodecylphosphocholine (DPC) micelles. The resulting perturbations in the structural-dynamics will be then correlated to functional effects observed in coupled enzyme assays in the presence of SERCA. From the insight of this work, the aim of this proposal is to rationally design new PLN mutant candidates for gene therapy and establish new principles for the successful design of therapeutic PLN analogs. PUBLIC HEALTH RELEVANCE: By controlling the function of an enyzme by acting on the structural dynamic of its'subunits, the results of this study aim to move from understanding the interplay of structural dynamics of controlling physiology at a molecular level.